Detection and identification of pathogenic microorganisms are indispensable for diagnoses of infectious diseases. However, detection and identification of a microorganism responsible for an infectious disease by cultivation is so time consuming that it is often not available for practical diagnosis and treatment of the disease. Therefore, diagnoses using immunological inspection methods have become more popular. In immunological inspection methods, proteins on the cell surface layers of microorganisms or antigenic substances produced in large amount during the process of proliferation are mainly detected, but these proteins, etc. are not always related to the microorganisms responsible for the diseases. This is because measuring the increased antibody titer in the serum of a patient for a specific pathogenic microorganism or the specific antigen thereof is measurement of a response to the infectious disease and is not confirmation of the microorganism per se.
Recently, investigation of the existence of a specified base sequence in a nucleic acid sample using a hybridization technique for nucleic acids has made it possible to specify a microorganism responsible for an infectious disease and diagnosis of the infectious disease before crisis. Thus, a method using a DNA probe for microorganism detection has been practiced. In this method, a single-stranded DNA having a base sequence complementary to the base sequence of a nucleic acid which is contained in a sample to be detected (the single-stranded DNA is referred to as "DNA probe") is utilized as a specifically reactive reagent. The existence of objective pathogenic bacteria can be judged by investigating the existence, in a sample, of a base sequence complementary to the base sequence of the DNA probe. Various practical methods have been proposed in, e.g., (1) Nippon Rinsho (Clinical Medicine in Japan), Vol. 47, 737-754, (1989), (2) E. M. Southern, "Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis", J. Mol. Biol., 98, pp503-517, (1975), and (3) J. Meinkoth & G. Wahl, "Hybridization of Nucleic Acids Immobilized on Solid Supports", Analytical Biochemistry, 138, pp267-284, (1984). One example of a method using the DNA probe is called dot hybridization. This method comprises attaching a single-stranded DNA (an SS-DNA) obtained by denaturation of a sample to a solid phase, allowing a radioisotope-labeled SS-DNA to act on the solid phase to form a hybrid between the labeled SS-DNA and the SS-DNA attached to the solid phase, removing the unreacted labeled SS-DNA, and measuring a radiation emitted from the solid phase (hereinafter referred to as "first prior art technique").
As a modification of the above method, there is a method of sandwich hybridization. This method makes it possible to reduce the background due to adsorption and hence is effective particularly when an impure sample is used. In this method, at least two DNA fragments derived from a target nucleic acid to be detected are used. One of the DNA fragments is attached to a solid phase and used as a capturing reagent. The other fragment is labeled as a reagent for detection and added to a solution containing the solid phase to which the capturing reagent is attached, together with a solubilized sample in a hybridizing solution. Then, the reagent for detection which has been bound to the solid phase by hybridization and the unreacted reagent for detection are separated from each other. When a base sequence homologous with both the reagents exists in the sample, the sequence is hybridized with both the capturing reagent and the reagent for detection. Whether the sequence has been hybridized or not can be known by measuring the label in the reagent for detection which has been bound to the capturing reagent in the solid phase through the sample. (This method will hereinafter be referred to as "second prior art technique".)
Further, JP, B, 3-78120 proposes a method using a restriction enzyme. The proposed method comprises bringing, in a solution, a single-stranded polynucleotide to be measured into contact with a solid phase combined with a single-stranded polynucleotide to which a labeling substance has been attached and which is capable of reacting with the single-stranded polynucleotide to be measured to form a double-stranded polynucleotide, thereby forming the double-stranded polynucleotide, allowing a restriction enzyme to act on the formed double-stranded polynucleotide to cleave this double-stranded polynucleotide, and measuring the labeling substance in the solution or solid phase (hereinafter referred to as "third prior art technique").
The above prior art methods however have the following problems.
The first and second prior art techniques are problematic, particularly when the amount of a nucleic acid to be detected is small, because the nucleic acid to be detected in the sample must finally be bound to the solid phase in either technique. Also, they require a large number of steps for completing measurement and, particularly, require the operation of separating the unreacted reagent in the solution for measuring the labeling substance in the reagent which has been hybridized with the nucleic acid to be detected. Further, immobilization of a sample slowly progresses because of a solid/liquid reaction and takes a long time. Thus, the first and second prior art technique are disadvantageous from the viewpoint of labor required for operations and time required for measurement.
In the third prior art technique, since a reagent capable of being treated beforehand is bound to a solid phase, immobilization of a sample is not necessary and the problems with the first and second prior arts are partly solved. However, the third prior art technique is disadvantageous in that a labeling substance is detected based on its concentration and hence detection sensitivity is not sufficient. For example, when an enzyme is used as the labeling substance, reliability of the measured values at low concentrations is poor because of not only limits in chemical amplification due to the enzyme reaction as high as about 104 at the maximum, but also influences of decomposition of the substrate, etc. The amplification due to the enzyme reaction is unstable because it is different depending on reaction conditions and is easily affected by coexisting substances. As a method of compensating for such a drawback, there is a method of amplifying DNA fragments of a sample. From the viewpoint of practical use, however, this method has problems of requiring a long time and causing contamination of a sample.
Additionally, in the third prior art technique, before cleaving each double strand formed by the reaction, completion of measurement also requires separation, in the solution, of the labeling substance which is in the unreacted reagent existing free in the solution and the labeling substance which has been attached to the solid phase by the reaction.